The present invention relates to a process for the separation of a body fluid from the particulate materials present in said fluid, in particular plasma from whole blood, to be used for a chemical analysis wherein a small amount of body fluid is contacted with a membrane or composite of membranes, said body fluid migrates into said membrane or composite whereas said particulate materials and non-migrated body fluid are retained and said migrated fluid is used for analysis purposes. Furthermore the present invention relates to a device for the separation of a body fluid from the particulate materials present in said fluid, in particular plasma from whole blood, comprising an inert solid support to which a membrane or composite of membranes is attached. Finally the invention relates to a testing kit for separation and chemical analysis of the components of a body fluid.
Although the present invention related to the separation of a body fluid from the particulate materials present therein and to a chemical analysis of said body fluid in general, such as lymph, urine and other body fluids the present invention will be further disclosed and illustrated by means of whole blood as a body fluid.
In view of diagnostic and therapy and for controlling of certain body functions there is since many years a need for analysis of body fluids in order to analyse the presence and/or concentration of a certain substance in said fluid. In particular blood is a frequently examined medium, which may lead to indications for several body functions and body processes. Since blood is a troubled medium because of the several particulate materials present therein, such as thrombocytes (blood platelets), erythrocytes (red blood cells) and leucocytes (white blood cells) whole blood is a troublesome medium for chemical analysis, since such particular materials do interfere with the usual analytical chemical techniques used for the determination of the presence and concentration of certain components. Accordingly several methods have been developed for the separation on the one hand of particulate materials and on the other hand of the plasma or serum from whole blood. For the somewhat larger amounts of blood (in the order of millimeters and more) many methods have been developed, which are mostly based on the centrifugation principle, wherein the blood cells are precipitated from the plasma or serum by means of the centrifugal force. However such methods usually do require complicated adjustments in order to recover the plasma or serum without the particulate materials. Another disadvantage of such techniques is that they require greater amounts of blood, which should be taken off by means of a venous puncture. Because of the above mentioned facts such methods are only used in the clinical chemical laboratory.
By the increasing demand for analysis data of body fluids, in particular blood, and by the increasing need for quick and simple analysis methods means are developed which only require a slight amount of blood, e.g. a drop of blood, obtained via a finger puncture. Such means exist usually in the form of a strip of an inert material, to which several layers are applied, which are able to achieve a separation between the particulate materials of the blood on the one hand and plasma or serum on the other hand, so that the particulate materials are retained in or on one or several of the upper layers, whereas the clear plasma is collected in the layers thereunder. In one or several of these layers reagents may be introduced. There reagents do simultaneously or after each other get in contact with the plasma and therefore they may react with the component to be determined. Because of this reaction a change in a physical property does occur, which change is a measure for the concentration of the substance to be determined, which change is observed visually or by means of suitable device, which leads to a semi-quantitative and quantitative result respectively.
Accordingly the U.S. Pat. No. 3,092,465 discloses the coating of a strongly sucking test paper, which is provided with a semi-permeable membrane for the determination of the blood sugar content. Herein blood is applied to the semi-permeable membrane, through which membrane only water and glucose pass but other bigger molecules such as hemoglobin or proteins are retained. Said glucose and water are taken up in the paper layer, which is provided with suitable reagents. The changing colour such obtained is then measured visually or remission- or reflection photometrically. This change in colour is observed through the semi-permeable membrane, which has therefore to be transparent and from which the disturbing particulate materials should be removed by means of washing or wiping. Such a method is only suitable for the determination of substance having small molecules.
Further it is known from German "Offenlegungsschrift" 1,598,153 to use a film which may swell up in water, which contains reagents. The components which are dissolved in the blood penetrate into the semi-permeable film, whereas the particulate materials are retained. This method has however similar disadvantages as the method which is disclosed in U.S. Pat. No. 3,092,465.
In order to improve such systems having semi-permeable membranes or films, several systems have been developed in order to separate particulate materials from blood to recover plasma or serum. Accordingly German "Offenlegungsschrift" 2,222,951 discloses a testing device for the determination of the enzyme activity in blood, wherein several different layers are arranged after each other, whereas one or several of these layers do function as a filter. Herein the upper two layers are porous glass fibre discs, which act as prefilter in order to retain particulate materials such as white blood cells, in order to prevent clogging of the underlying membrane filter disc. The membrane filter disc acts as a filter for removing the red blood cells and other particles. Finally under said membrane filter disc there is provided a disc of cellulose acetate. Each of the separate discs provide distinctive test zones. The colour change which is obtained by such analysis method is observed visually. The disadvantage of such method is that the plasma only very slowly and in slight amounts may penetrate into the membrane filter, whereas this membrane filter may be clogged easily, which means that said plasma slowly and unequally penetrates into the reaction layer. Furthermore German "Offenlegungsschrift" 2,922,958 discloses a multi layer element, having at least four layers consisting of an upper filter layer, a water impermeable layer thereunder having one or more orifices, then an expanding layer and finally a reagent layer. As filter layer use is made of one or several membrane filters, whereas the porous expanding layer may also be a membrane filter. It is stated that the expanding layer is weak and fragile (see page 15, sub 5) so that this layer should be sandwiched between other layers in order to prevent problems during handling. Such a multi layer element has similar disadvantages as mentioned in the German "Offenlegungsschrift" 2,222,951.
The above discussed methods wherein on the one hand use is made of semi-permeable membranes and on the other hand use is made of membrane filters (porous membranes) did not lead to fully satisfactory results. A further development of such methods is disclosed in the European patent application 45476, wherein it is suggested to apply a layer of glass fibres in the form of a disc onto the known testing devices as cover. The glass fibre disc retains the particulate materials from the blood and enables to pass serum or plasma into the layers thereunder. As disadvantages of such a system are mentioned (see European patent application 133895, page 3) the relatively high dead volume of this glass fibre disc as well as the fact that such a disc is not able to retain the erythrocytes completely, so that blood colour penetrates into the reaction zone which disturbs the reaction. Therefore in the last mentioned European patent application a substrate is suggested which should be contacted with blood and which substrate together with blood should be passed through a specific filter disc, so that the separated plasma may be recovered in the layers thereunder.
Finally according to the methods disclosed in the European patent application Nrs. 0154839 and 0175990 the blood sample is applied to that side of the membrane where the pore size is the smallest (the so-called smooth surface of the membrane). This has as serious disadvantage that the small pores are clogged by the blood particles such as thrombocytes, erythrocytes, leucocytes and coagulates thereof, resulting in the fact that only very slight amounts of plasma penetrate into the membrane. This means that the reaction time is too long with the risk that the determination is inaccurate. (In connection with the above mentioned disadvantages reference is made to European patent application No. 0045476 in the name of Boehringer Mannheim GmbH, page 2, lines 14 through 20).
Another disadvantage is that the blood has to be wiped off from the smooth surface of the membrane, whereby damaging of the erythrocytes is practically unavoidable, which means that the desired colour reaction is disturbed.
In conclusion there are several methods and testing devices available, which are also known as quick diagnostics, which may be used for the analysis of a slight amount, e.g., a drop, of whole blood. However in case of the use of semi-permeable membranes there is not obtained the total plasma or serum, but only the components to be determined with a small molecule size, whereas in using membrane filters usually complicated provisions should be taken in order to safeguard a proper process. Furthermore it should be noted that frequently cloggings occur and that the plasma or serum only slowly passes through such membranes. The recent developments in this art make use of other filter discs than membrane filters. Except the above mentioned disadvantages and shortcomings each of the above mentioned methods relate to "dry" analysis methods. This means that the result, usually a colour change, should be evaluated visually or by means of reflection photometric. A visual method is however not quantitative, whereas a reflection photometric method requires a relatively expensive measuring device and make high demands of the test systems. Further the known test systems are usually provided with swellable reagent layers, so that it is often difficult to obtain a reproduceable result, since a drop of blood, in particular when said drop of blood should be applied on such a test system, mostly does not lead to a reproducable result. Furthermore the long time period which is required for the plasma to penetrate in such systems may result in the fact that the blood begins to coagulate, resulting in clogging of the entire system.
Therefore there is a need for a simple and cheap method and device for the chemical analysis of components of body fluids, wherein use is made of a drop or a small amount of such a liquid, wherein the above mentioned disadvantages are eliminated. Furthermore there is also need for a quick, simple and accurate "wet" analysis of such fluids, wherein relatively cheap analysis apparatus may be used, such as e.g., colorimeters or photometers, which means that such a method is accessible for routine examinations, not only in clinical laboratories, but also by general practitioners and so on.